MicroRNA-based approach to treating malignant pleural mesothelioma

ABSTRACT

The invention relates to microRNA mimics, corresponding to the miR-15/107 family, and to methodology for using microRNA mimics to treat malignant pleural mesothelioma (MPM) by restoring regulation of the expression of target genes of the miR-15/107 family in MPM tumor cells.

SEQUENCE LISTING

The present application contains a sequence listing, which has beensubmitted in ASCII format via EFS-Web and which is hereby incorporatedby reference in its entirety. The ASCII copy, created on Jun. 20, 2013,is named 104481-0102_SL.txt and is 6,399 bytes in size.

BACKGROUND OF THE INVENTION

1. Technical Field

The invention relates generally to the field of molecular biology andcancer. More specifically, the invention relates to microRNA mimicscorresponding to the miR-15/107 family and related methods of usingmicroRNA mimics to treat malignant pleural mesothelioma (MPM) byrestoring the regulation of expression of target genes of the miR-15/107family in MPM tumor cells.

2. Background

Malignant pleural mesothelioma is an almost invariably fatal cancer forwhich few treatments are available. New therapies are urgently needed,and dysregulated microRNA expression provides a source of noveltherapeutic targets.

MicroRNAs are transcribed by RNA polymerase II (pol II) or RNApolymerase III (pol III). See Qi et al. (2006) Cellular & MolecularImmunology, Vol. 3: 411-19. They arise from initial transcripts, termedprimary microRNA transcripts (pri-microRNAs), which generally areseveral thousand bases long. Pri-microRNAs are processed in the nucleusby the RNase Drosha into about 70- to about 100-nucleotidehairpin-shaped precursors (pre-microRNAs). Following transport, to thecytoplasm, the hairpin pre-microRNA is further processed by Dicer toproduce a double-stranded mature microRNA. The mature microRNA strand isthen incorporated into the RNA-induced silencing complex (RISC), whereit associates with its target mRNAs by base-pair complementarity. In therelatively rare cases in which a microRNA base pairs perfectly with anmRNA target, it promotes mRNA degradation. More commonly, microRNAs formimperfect heterduplexes with target mRNAs, affecting either mRNAstability of inhibiting mRNA translation.

Multiple studies have profiled mRNA gene expression in MPM to identifypotential targets, and more recently, microRNA expression profiles havebeen generated, initially for diagnostic purposes using samples derivedfrom normal and tumor cell lines, MPM tumors and pooled normalpericardium, or MPM and lung cancer. They also have been generated forprognostic purposes, i.e., within MPM tumors of differentclassification. See, e.g., Busacca et al., Am. J. Respir. Cell. Mol.Biol. 42: 312-19 (2010). However, none have made an extensive comparisonbetween MPM tumors and normal pleural tissue.

Further, while relatively easy to use in vitro, microRNA mimicstypically suffer, in terms of in vivo efficacy, due to two problems: (1)poor activity (including low RISC incorporation and off-target effects)and (2) inefficient delivery (related to stability andspecific/selective distribution to the site of action).

As mentioned, multiple studies have profiled gene expression in MPM.This has been with the aim of understanding the disease process as wellas to identify potential targets. These studies have characterized ageneral upregulation of metabolic and cell cycle genes in MPM, withadditional changes in apoptotic genes associated with an alteredapoptotic response linked to resistance to chemotherapy. To date, thesestudies have yet to reveal the overarching mechanism of genetic controlof the phenotypes common to MPM tumors. However, as microRNAs areconsidered global modulators of gene expression, downregulation ofexpression of microRNAs represent a potential explanation for theupregulation of families of genes (i.e., loss of microRNA expressioncauses target gene upregulation).

SUMMARY OF THE INVENTION

The invention is based in part on the identification of a family ofmicroRNAs that are down-regulated in MPM tumor samples as compared withnormal pleural tissue from unaffected individuals. In particular, theinventors discovered a marked down-regulation in expression of themiR-15/107 family of microRNAs in MPM tissue.

Accordingly, one aspect of the present invention relates to adouble-stranded microRNA mimic that is useful for the treatment of MPM.Such a miRNA mimic of the invention comprises:

(1) a mature sequence corresponding to a miR-15/107 family member andthat contains AGCAGC at positions 2-7 or 1-6 at the 5′ end; and (2) apassenger strand, which can be inactivated by chemical modification. Themature sequence can comprise a sequence selected from the groupconsisting of SEQ ID NOS: 11-14. The passenger strand can comprise asequence selected from the group consisting of SEQ ID NOS: 15-18.

In accordance with another aspect, the invention provides a method fortreating MPM in a subject suffering from the disease. The methodcomprises administering an effective amount of a double-stranded miRNAmimic, as described above, where such administration mimics endogenousexpression of the miR-15/107 family, thereby restoring regulation of theexpression of target genes of the miR-15/107 family in the subject. Theadministration preferably is effected using an intact, bacteriallyderived minicell for delivery of the miRNA mimic. Pursuant to theinventive methodology, the step of administering microRNA mimiccomprises simultaneous or serial co-administration of an adjunctanti-cancer therapy to the subject.

In another aspect of the invention, a method is provided for increasingsensitivity of a MPM cancer cell to the cytotoxic effects of ananti-cancer therapy. The method comprises administering to the cell atleast one miRNA mimic as described above, such that the sensitivity ofthe MPM cancer cell is increased.

These and other aspects of this invention are further described below.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended figures. These figures form a part of thespecification. They illustrate particular embodiments of the inventionand, hence, are not limiting of it.

FIG. 1 depicts the expression of the miR-15/107 family which isdownregulated in MPM cell lines and tumors.

FIG. 2 illustrates that continuous chrysotile exposure reducesmiR-15/107 family microRNA expression in MeT-5A immortalized mesothelialcells

FIG. 3 depicts that replacing the miR-15/16 family leads to growthinhibition of MPM cells in vitro.

FIG. 4 shows that mimics comprising a sequence corresponding to themiR-15/107 consensus are more effective than native miR-16 in inhibitingMPM cell proliferation

FIG. 5 illustrates that transfection with miR-16 downregulates targetgenes.

FIG. 6 depicts the effects of miR-16 on gemcitabine and pemetrexedtoxicity in MPM cells.

FIG. 7 depicts the effects of miR-16 replacement in MPM in vivo,delivered as ^(EGFR)minicells_(miR-16) (bispecific antibody targeted,miR-16-packaged minicells where the tumor cell targeting sequence in thebispecific antibody is directed to EGFR).

DETAILED DESCRIPTION OF THE INVENTION

As noted, a key aspect of the invention is the identification of afamily of microRNAs that are down-regulated in MPM tumor samples ascompared with normal pleural tissue from unaffected individuals. Thus, amarked down-regulation of the expression of the miR-15/107 family ofmicroRNAs occurs in MPM tissue.

In exemplary embodiments, therefore, the present invention relates tothe design, synthesis, construction, composition, characterization anduse of therapeutic microRNAs corresponding to the miR-15/107 family fortreating MPM. More specifically, the invention is directed to microRNAmimics that act to restore expression of the miR-15/107 family in MPMtumor cells by mimicking the activity of the endogenous members of themiR-15/107 family, thereby re-establishing control of MPM cell growth.The microRNA mimics can therefore be used in a replacement therapyapproach to restore expression of the miR-15/107 family in MPM cells.

In further exemplary embodiments, the microRNA mimics are modified toimprove stability, reduce off-target effects and increase activity.Additional embodiments relate to the use of a minicell to deliver themicroRNA mimics. In another aspect of the invention, microRNA mimicsoperate to enhance the efficacy of other clinically used drugs for thetreatment of MPM.

The miR-15/107 Family

The miR-15/107 family was identified previously and is characterized asa microRNA super family in which each member contains the sequenceAGCAGC starting at either the first or second nucleotide from the 5′ endof the mature microRNA strand. It is believed that the miR-15/107 familyis involved with the regulation of numerous cell activities thatrepresent intervention points for cancer therapy and for therapy ofother diseases and disorders. See Finnerty et al. (2010) J. Mol. Biol.,Vol. 402: 491-509, the contents of which are incorporated here byreference in its entirety. For instance, the miR-15/107 family isbelieved to be involved in regulating gene expression relating to celldivision, metabolism, stress response and angiogenesis in vertebratespecies, as well as human cancers, cardiovascular disease andneurodegenerative diseases.

The search for novel approaches to developing cancer therapies oftenbegins with attempts to identify differences between normal and tumortissue to enable specific or selective targeting of tumor cells. For MPMthis has proved difficult as many changes in gene expression, whilespecific to the tumor cells, are either unable to be targeted (i.e.,non-druggable) or provide a therapeutic window that is too narrow (i.e.,normal cells are affected). Furthermore, target expression is not alwayscorrelated with treatment success. While previous studies have indicatedthat a number of microRNAs have tumor-suppressor or oncogenic functionsin MPM, these have not been observed to occur frequently in a largeproportion of tumors or cell lines. In this context, the discovery thatthe entire miR-15/107 family is downregulated in all MPM cell lines andtumors analyzed (FIG. 1) provides a strong rationale for targeting thesechanges as a treatment approach. Together with data showingasbestos-induced downregulation of the miR-15/107 family of microRNAs(FIG. 2), this evidences an important causative role of these changes inMPM biology. In this regard, the identification of downregulation of themiR-15/107 family is significant for at least two reasons: first, it ishighly specific for MPM (change is present in all samples, FIG. 1);second, it has no effect on normal mesothelial cells (FIG. 3). Inaddition, using a microRNA as a therapeutic entity for MPM provides theability to correct, with a single agent, multiple aberrantly expressedgenes (FIG. 5) responsible for increased proliferation and drugresistance (FIG. 6). To formulate microRNAs that are capable ofrestoring expression of the miR-15/107 family, the inventors consideredcharacteristics such as sequence similarity in order to prepare aneffective microRNA therapeutic approach.

The miR-15/107 family shares a common seed sequence, and thus the mRNAtargets of each member overlap significantly. As used herein, the term“microRNA family” refers to a group of miRNA species that share identityacross at least 6 consecutive nucleotides, also referred to as the “seedsequence,” as described in Brennecke, J. et al., PloS biol 3 (3):pe85(2005). As used herein, the term of “seed sequence” is used to denotenucleotides at positions 1-6, 1-7, 2-7, or 2-8 of a mature miRNAsequence. The microRNA seed sequence typically is located at the 5′ endof the miRNA. As used herein, the term “mature sequence” refers to thestrand of a fully processed microRNA that enters RISC.

Accordingly, for the purposes of the present invention the miR-15/107family is comprised of ten sequences as follows:

miR 15a-5p (SEQ ID NO: 1 uagcagcacauaaugguuugug, MIMAT0000068);miR-15b-5p (SEQ ID NO: 2 uagcagcacaucaugguuuaca, MIMAT0000417);miR-16-5p (SEQ ID NO: 3 uagcagcacguaaauauuggcg, MIMAT0000069);miR-195-5p  (SEQ ID NO: 4 uagcagcacagaaauauuggc, MIMAT0000461);miR-424-5p (SEQ ID NO: 5 cagcagcaauucauguuuugaa, MIMAT0001341);miR-497-5p (SEQ ID NO: 6 cagcagcacacugugguuugu, MIMAT002820); miR-503-5p(SEQ ID NO: 7 uagcagcgggaacaguucugcag, MIMAT0002874); miR-646-5p(SEQ ID NO: 8 aagcagcugccucugaggc, MIMAT0003316); miR-103a-3p(SEQ ID NO: 9 agcagcauuguacagggcuauga, MIMAT0000101); and miR-107(SEQ ID NO: 10 agagcauuguacagggcuauca, MIMAT0000104).

To control all targets of the miR-15/107 family effectively, in theoryall ten members would need to be reintroduced to MPM cells usingmicroRNA mimics specific to each sequence listed above. Yet, this is notan efficient approach to clinical treatment. Instead, the inventionprovides a microRNA mimic approach in which a consensus sequence of theentire miR-15/107 family has been designed that operates as a mimic toperform the functions of the endogenous miR-15/107 family, therebyrestoring expression of the miR-15/107 family in MPM cells (FIG. 3). Asused in this description, the phrase “consensus sequence” refers to anucleotide sequence that shares high sequence, structural and/orfunctional identity among a group of sequences. In this regard, amicroRNA mimic comprising a consensus sequence is capable of mimickingthe functions of the entire miR-15/107 family. The design of a consensussequence of the entire miR-15/107 family therefore affords the advantageof maximizing the number of desired targets (i.e., the ten miR-15/107family members) for which endogenous expression can be mimicked, whileat the same time limiting the number of mimics to a single sequenceentity.

Accordingly, in specific embodiments, the microRNA mimics are used as aform of replacement therapy to treat MPM cells, wherein the microRNAmimics are capable of performing the functions of the miR-15/107 family,thereby restoring expression of the miR-15/107 family. As such, in thecontext of this disclosure, the term “restoring expression” refers tothe restoration of expression of the miR-15/107 family through the useof microRNA mimics that are capable of mimicking the functions of theendogenous miR-15/107 family.

microRNA Mimics

As used herein, the term “microRNA mimic” refers to synthetic non-codingRNAs that are capable of entering the RNAi pathway and regulating geneexpression. As used herein, “synthetic microRNA” refers to any type ofRNA sequence, other than endogenous microRNA. MicroRNA mimics imitatethe function of endogeneous microRNAs and can be designed as mature,double-stranded molecules or mimic precursors (e.g., pri- orpre-microRNAs). MicroRNA mimics can be comprised of modified orunmodified RNA, DNA, RNA-DNA hybrids or alternative nucleic acidchemistries.

As disclosed above, the miR-15/107 family comprises ten microRNAs thatshare the sequence 5′-AGCAGC-3′ at the 5′-terminal end of the active(guide) strand. The fact that all members of the miR-15/107 family havethe same seed sequence provides an opportunity to correct globalregulation of gene expression with a single microRNA mimic. Accordingly,the invention provides microRNA mimics corresponding to the miR-15/107family which comprise a consensus sequence, wherein the microRNA mimicsare capable of mimicking the endogenous activity of the entiremiR-15/107 family. Therefore, restoration of microRNA expression isachieved through the use of these microRNA mimics.

Exemplary consensus sequences of the invention are as follows:

miR-15/107-consensus-1 (SEQ ID NO: 11 UAGCAGCACAUAAUGGUUUGCG);miR-15/107-consensus-2 (SEQ ID NO: 12 UAGCAGCACAUAAUGGUUUGCGGA;miR-15/107-consensus-3 (SEQ ID NO: 13 UAGCAGCACAUAAUGGUUUGCU); andmiR 15/107-consensus-4 (SEQ ID NO: 14 UAGCAGCACAGUAUGGUUUGCG).miR-15/107-complement-1 (SEQ ID NO: 15 CGCAAACCAUUAUGUGCUGCUA);miR-15/107-complement-2 (SEQ ID NO: 16 UCCGCAAACCAUUAUGUGCUGCUA);miR-15/107-complement-3 (SEQ ID NO: 17 AGCAAACCAUUAUGUGCUGCUA);miR-15/107-complement-4 (SEQ ID NO: 18 CGCAAACCAUACUGUGCUGCUA);miR-15/107-complement-1a (SEQ ID NO: 19 CGCAAACCAUUAUGUGCUGCUU);miR-15/107-complement-2a (SEQ ID NO: 20 UCCGCAAACCAUUAUGUGCUGCUU);miR-15/107-complement-3a (SEQ ID NO: 21 AGCAAACCAUUAUGUGCUGCUU);miR-15/107-complement-4a (SEQ ID NO: 22 CGCAAACCAUACUGUGCUGCUU);miR-15/107-complement-1b (SEQ ID NO: 23 CGCAAACCAUUAUGUGCUGCUUUA);miR-15/107-complement-2b (SEQ ID NO: 24 UCCGCAAACCAUUAUGUGCUGCUUUA);miR-15/107-complement-3b (SEQ ID NO: 25 AGCAAACCAUUAUGUGCUGCUUUA);miR-15/107-complement-4b (SEQ ID NO: 26 CGCAAACCAUACUGUGCUGCUUUA);miR-15/107-complement-1c (SEQ ID NO: 27 CGCAAACCAUUAUGUGCUGCUUUA);miR-15/107-complement-2c (SEQ ID NO: 28 UCCGCAAACCAUUAUUGUGCUGCUUUA);miR-15/107-complement-3c (SEQ ID NO: 29 AGCAAACCAUUAUUGUGCUGCUUUA); andmiR-15/107-complement-4c (SEQ ID NO: 30 CGCAAACCAUACUUGUGCUGCUUUA).

A preferred embodiment of the invention comprises a synthetic consensusmicroRNA (“guide”) sequence in full or in part (SEQ IDs NO: 11-14),together with the complementary sequence as a passenger strand (SEQ IDsNO: 15-18). A double-stranded RNA mimic in which terminal mismatchesand/or internal bulges are incorporated between the guide and passengerstrand, which are introduced to increase RISC loading, is alsocontemplated by the invention (see SEQ ID NOs: 21-30). Other variationsof the sequence corresponding to the consensus sequence of all familymembers, where the seed sequence AGCAGC is present within the first 7nucleotides of the guide strand, i.e., positions 1-6 or positions 2-7,are also contemplated. Those skilled in the art will understand thatother variations can promote RISC loading to increase activity. By wayof example, these include a one or two nucleotide overhang at the 3′ endof the guide strand; a DNA nucleotide (or other chemical modification)at the 3′ end of the passenger strand.

Chemical Modifications

Generally, microRNA mimics have been found to be inefficient inoperative use. In this regard, to improve efficiency the presentinvention employs a microRNA mimic comprising a structurally andchemically modified double-stranded RNA. In exemplary embodiments, inorder to overcome the limitations of microRNA mimics, non-toxic chemicalmodifications to the mimic sequence have been introduced to improvestability, reduce off-target effects and increase activity.

In one embodiment, the microRNA mimic includes an RNA duplex comprisingthe mature microRNA sequence and a passenger strand. In one aspect, thepassenger strand is structurally and chemically modified to enable theretention of activity of the duplex mimic while inactivating thepassenger strand, thereby reducing off-target effects. In a furtheraspect, chemical modification inhibits nuclease activity, therebyincreasing stability.

In particular embodiments, the microRNA mimics of the inventioncontemplate the use of nucleotides that are modified to enhance theiractivities. Such nucleotides include those that are at the 5′ or 3′terminus of the RNA as well as those that are internal within themolecule. Modified nucleotides used in the complementary strands ofmicroRNAs either block the 5′OH or phosphate of the RNA or introduceinternal sugar modifications that prevent uptake and activity of theinactive strand of the microRNA. Modifications for the microRNAinhibitors include internal sugar modifications that enhancehybridization as well as stabilize the molecules in cells and terminalmodifications that further stabilize the nucleic acids in cells. Furthercontemplated are modifications that can be detected by microscopy orother methods to identify cells that contain the microRNAs.

In other aspects, modifications may be made to the sequence of amicroRNA or a pre-microRNA without disrupting microRNA activity. As usedherein, the term “functional variant” of a microRNA sequence refers toan oligonucleotide sequence that varies from the natural microRNAsequence, but retains one or more functional characteristics of themicroRNA (e.g. enhancement of cancer cell susceptibility tochemotherapeutic agents, cancer cell proliferation inhibition, inductionof cancer cell apoptosis, specific microRNA target inhibition). In someembodiments, a functional variant of a microRNA sequence retains all ofthe functional characteristics of the microRNA. In certain embodiments,a functional variant of a microRNA has a nucleobase sequence that is aleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the microRNA or precursor thereof overa region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100 or more nucleobases, or that the functional varianthybridizes to the complement of the microRNA or precursor thereof understringent hybridization conditions. Accordingly, in certain embodimentsthe nucleobase sequence of a functional variant may be capable ofhybridizing to one or more target sequences of the microRNA.

In some embodiments, the complementary strand is modified so that achemical group other than a phosphate or hydroxyl is at its 5′ terminus.The presence of the 5′ modification apparently eliminates uptake of thecomplementary strand and subsequently favors uptake of the active strandby the microRNA protein complex. The 5′ modification can be any of avariety of molecules known in the art, including NH₂, NHCOCH₃, andbiotin.

In another embodiment, the uptake of the complementary strand by themicroRNA pathway is reduced by incorporating nucleotides with sugarmodifications in the first 2-6 nucleotides of the complementary strand.It should be noted that such sugar modifications can be combined withthe 5′ terminal modifications described above to further enhancemicroRNA activity. Sugar modifications contemplated in microRNA mimicsinclude, but are not limited to, a sugar substitute group selected from:F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. In some embodiments, these groups may be chosen from:O(CH₂)_(x)OCH₃, O((CH₂)_(x)O)_(y)CH₃, O(CH₂)_(x)NH₂, O(CH₂)_(x)CH₃,O(CH₂)_(x)ONH₂ and O(CH₂)_(x)ON((CH₂)_(x)CH₃)₂, where x and y are from 1to 10.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the purpose of a microRNA mimic. Sucholigomeric compounds are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified oligonucleotides. As such, all sucholigomeric compounds are contemplated to be encompassed by thisinvention so long as they function effectively to mimic the structure orfunction of a desired RNA oligonucleotide strand corresponding to themiR-15/107 family.

In some embodiments, the complementary strand is designed so thatnucleotides in the 3′ end of the complementary strand are notcomplementary to the active strand. This results in double-strandedhybrid RNAs that are stable at the 3′ end of the active strand butrelatively unstable at the 5′ end of the active strand. This differencein stability enhances the uptake of the active strand by the microRNApathway, while reducing uptake of the complementary strand, therebyenhancing microRNA activity.

Other modifications contemplated in the practice of the invention can befound in US Patent Pub. No. 2012/0259001, which is incorporated hereinby reference in its entirety.

In some embodiments, microRNA sequences of the invention may beassociated with a second RNA sequence that may be located on the sameRNA molecule or on a separate RNA molecule as the microRNA sequence. Insuch cases, the microRNA sequence may be referred to as the activestrand, while the encoded RNA sequence, which is at least partiallycomplementary to the microRNA sequence, may be referred to as thecomplementary strand. The active and complementary strands may behybridized to generate a double-stranded RNA that is similar to anaturally occurring microRNA precursor. The activity of a microRNA maybe optimized by maximizing uptake of the active strand and minimizinguptake of the complementary strand by the microRNA protein complex thatregulates gene translation. This can be done through modification and/ordesign of the complementary strand, for instance.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. In some embodiments, microRNAcompositions of the invention are chemically synthesized.

A preferred embodiment has a particular set of such modifications. Theseare chemical modification of 2 to 6 nucleotides at each end of thepassenger strand with 2′O-methyl-modified sugars, and include anycombination of 2, 3, 4, 5 or 6 modified nucleotides at the 5′ end of thepassenger strand with 2, 3, 4, 5 or 6 modified nucleotides at the 3′ endof the passenger strand. Alternative chemical modification strategiesimparting similar functionality will be apparent to those skilled in theart.

Method of Administration

MicroRNA mimics can be administered to a subject by any means suitablefor delivering these compounds to cancer cells of the subject. Forexample, the microRNA mimics can be administered by methods suitable totransfect cells of the subject with the mimics, or with nucleic acidscomprising sequences encoding these compounds. In one embodiment, thecells are transfected with a plasmid or viral vector comprisingsequences encoding a microRNA mimic.

In one particular embodiment of the invention, to circumvent theproblems associated with inefficient delivery in vivo, a mimic accordingto the invention preferably is delivered via the “EnGeneIC DeliveryVehicle” system developed by EnGeneIC Molecular Delivery Ltd. (Sydney),which is based on the use of intact, bacterially derived minicells. TheEDV system is described, for example, in published internationalapplications WO 2006/021894 and WO 2009/027830, the respective contentsof which are incorporated here by reference.

In an exemplary embodiment, the microRNA mimics described here aredelivered using intact, bacterially derived minicells or EnGeneICdelivery vehicles (EDVs). These EDVs are delivered specifically totarget tissues using bispecific antibodies. One arm of such an antibodyhas specificity for the target tissue, while the other has specificityfor the EDV. The antibody brings the EDVs to the target cell surface,and then the EDV is brought into the cell by endocytosis. After uptakeinto the tumor cell there is a release of the EDV contents, i.e., themicroRNA mimic(s). For an antibody in this regard, specificity againstany cell surface marker for MPM could be used in accordance with theinvention. Thus, illustrative of such specificity suitable for abispecific antibody in the present context could be a specificity tohuman mesothelin, expressed on 100% of epithelioid mesotheliomas, forwhich therapeutic antibodies are in development (see Kelly et al., Mol.Cancer. Ther. 11: 517-22 (2012)), or to intelectin-1, which is expressedspecifically in MPM and gastrointestinal goblet cells (see Washimi etal., PLoS One 7: e39889 (2012)).

Other methods of administering nucleic acids are well known in the art.In particular, the routes of administration already in use for nucleicacid therapeutics, along with formulations in current use, providepreferred routes of administration and formulation for the nucleicacids. Nucleic acid compositions can be administered by a number ofroutes including, but not limited to: oral, intravenous, intrapleural,intraperitoneal, intramuscular, transdermal, subcutaneous, topical,sublingual, or rectal means.

Nucleic acids can also be administered via liposomes or nanoparticles.Such administration routes and appropriate formulations are generallyknown to those of skill in the art. Administration of the formulationsdescribed herein may be accomplished by any acceptable method thatallows the microRNA or nucleic acid encoding the microRNA to reach itstarget. The particular mode selected will depend of course, uponexemplary factors such as the particular formulation, the severity ofthe state of the subject being treated, and the dosage required fortherapeutic efficacy. As generally used herein, an “effective amount” ofa nucleic acid is the amount that is able to treat one or more symptomsof cancer or related disease, reverse the progression of one or moresymptoms of cancer or related disease, halt the progression of one ormore symptoms of cancer or related disease, or prevent the occurrence ofone or more symptoms of cancer or related disease in a subject to whomthe formulation is administered, as compared to a matched subject notreceiving the compound or therapeutic agent. The actual effectiveamounts of drug can vary according to the specific drug or combinationthereof being utilized, the particular composition formulated, the modeof administration, and the age, weight, condition of the subject, andseverity of the symptoms or condition being treated.

Other delivery systems suitable include but are not limited totime-release, delayed release, sustained release, or controlled releasedelivery systems. Such systems may avoid repeated administrations inmany cases, increasing convenience to the subject and the physician.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include, for example, polymer-basedsystems such as polylactic and/or polyglycolic acids, polyanhydrides,polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and/or combinations of these.

Pharmaceutical compositions of the invention containing microRNA mimicscan also comprise conventional pharmaceutical excipients and/oradditives. Suitable pharmaceutical excipients include stabilizers,antioxidants, osmolality adjusting agents, buffers, and pH adjustingagents. Suitable additives include, e.g., physiologically biocompatiblebuffers (e.g., tromethamine hydrochloride), additions of chelants (e.g.,DTPA or DTPA-bisamide) or calcium chelate complexes (e.g., calcium DTPA,CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts(e.g., calcium chloride, calcium ascorbate, calcium gluconate or calciumlactate).

Dosing

As the relationship between loss of microRNA expression and MPM isconsistent across the samples, one aspect of the invention is directedto microRNA replacement in MPM tumor cells. In this aspect, based oninitial animal experiments, it is contemplated that the progression ofMPM will be halted by treatment with the miR-containing miRs of theinvention. In further aspects, it is contemplated that multiple dosesover a continued time frame can be administered to the subject. In someaspects, although one dose per week may have a suitably efficaciouseffect, some subjects may demonstrate more markedly efficacious effectsin response to increasing frequency and/or increasing dosage.

In another embodiment of the invention, chronic dosing will be able toachieve MPM disease control. In yet other embodiments, it iscontemplated that MPM tumor cells receiving microRNA mimics will enterpermanent growth arrest. In this regard, permanent growth arrest can beattributed to the mechanism of action of the microRNA mimics(downregulation of cell cycle- and metabolism-promoting genes,anti-apoptotic pro-survival genes, and drug resistance genes) andphenotypic consequences (cell cycle arrest). Continued treatment thus iscontemplated, in accordance with one aspect of the invention, to inducedisease control or maintenance (i.e., stable disease). Pursuant toaspect of the invention, tumor regression (i.e., a partial/completeresponse) is contemplated where the MPM tumors enter permanent cellgrowth arrest.

Furthermore, as discussed in further detail below, the microRNA mimicsof the invention further function to sensitize cells to conventionalcancer therapies such as chemotherapy and radiation. In this aspect, itis contemplated that combining the mimics with chemotherapy will provideadditional therapeutic effects.

With all current treatments for MPM, response to therapy is invariablyfollowed by relapse. Thus, in yet another aspect of the invention it iscontemplated that tumors will retain this expression profile uponrelapse in view of the relationship between microRNA expression and MPM.In this regard, a relapse as described will allow re-treatment with thesame microRNA mimics disclosed herein. In this embodiment, long-termtumor suppression changes the nature of disease and potentially changesMPM into a form of chronic disease.

In other aspects, dosages for a particular subject can be determined byone of ordinary skill in the art using conventional considerations,(e.g., by means of an appropriate, conventional pharmacologicalprotocol). A physician may, for example, prescribe a relatively low doseat first, subsequently increasing the dose until an appropriate responseis obtained. The dose administered to a subject is sufficient to effecta beneficial therapeutic response in the subject over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of themicroRNA employed and the condition of the subject, as well as the bodyweight or surface area of the subject to be treated. The size of thedose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, and formulation, in a particular subject.

Combination Therapy

The microRNA mimics described herein can supplement treatment conditionsby any known conventional therapy, including, but not limited to,antibody administration, vaccine administration, administration ofcytotoxic agents, natural amino acid polypeptides, nucleic acids,nucleotide analogues, and biologic response modifiers. Two or morecombined compounds may be used together or sequentially. For example,the microRNA mimics can also be administered in therapeuticallyeffective amounts as a portion of an anticancer cocktail. An anti-cancercocktail is a mixture of the microRNA mimic(s) with one or moreanti-cancer drugs in addition to a pharmaceutically acceptable carrierfor delivery. The use of anti-cancer cocktails as a cancer treatment isroutine.

In one embodiment, it is envisioned to use a microRNA mimic incombination with other therapeutic modalities. Thus, in addition to themicroRNA therapies described above, one may also provide to the subjectmore “standard” therapies such as, but not limited to, conventionalcancer therapeutic agents.

Anti-cancer drugs that are well known in the art and can be used as atreatment in combination with the nucleic acids described hereininclude, but are not limited to: actinomycin D, aminoglutethimide,asparaginase, bleomycin, busulfan, carboplatin, carmustine,chlorambucil, cisplatin (cis-DDP), cyclophosphamide, cytarabine HCl(cytosine arabinoside), dacarbazine, factinomycin, daunorubicin HCl,doxorubicin HCl, Estramustine phosphate sodium, etoposide (VP 16-213),floxuridine, 5-fluorouracil (5-FU), flutamide, hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon apha-2a, interferon alpha-2b,leuprolide acetate (LHRH-releasing factor analog), lomustine,mechlorethamine HCl (nitrogen mustard), melphalan, mercaptopurine,mesna, methotrexate (MTX), mitomycin, mitoxantrone hcl, octreotide,plicamycin, procarbazine hcl, streptozocin, tamoxifen citrate,thioguanine, thiotepa, vinblastine sulfate, vincristine sulfate,amsacrine, azacitidine, hexamethylmelamine, interleukin-2, mitoguazone,pentostatin, semustine, teniposide, and vindesine sulfate.

Chemotherapeutic agents, for example, can be agents that directlycross-link DNA, agents that intercalate into DNA, and agents that leadto chromosomal and mitotic aberrations by affecting nucleic acidsynthesis. Agents that directly cross-link nucleic acids, specificallyDNA, are envisaged and are shown herein, to eventuate DNA damage leadingto a synergistic antineoplastic combination. Agents such as cisplatin,and other DNA alkylating agents may be used. Agents that damage DNA alsoinclude compounds that interfere with DNA replication, mitosis, andchromosomal segregation. Examples of these compounds include adriamycin(also known as doxorubicin), VP-16, also known as etoposide, verapamil,podophyllotoxin, and the like. Exemplary chemotherapeutics include atleast I) antibiotics, such as doxorubicin, daunorubicin, mitomycin,Actinomycin D; 2) platinum-based agents, such as cisplatin; 3) plantalkaloids, such as taxol and vincristine, vinblastine; 4) alkylatingagents, such as carmustine, melphalan, cyclophosphamide, chlorambucil,busulfan, and lomustine.

In exemplary embodiments of the invention, the microRNA mimic(s) can beadministered as a single agent but can also be used in combination withother drugs, e.g., pemetrexed, cisplatin (or carboplatin), andgemcitabine, etc.

Combination therapies may be achieved by contacting MPM tumor cells witha single composition or a pharmacological formulation that includes oneor more microRNA mimics and a second cancer therapeutic agent, or bycontacting the tumor cell with two distinct compositions orformulations, at the same time, wherein one composition includes one ormore microRNA mimics and the other includes the second cancertherapeutic agent. Alternatively, administration of one or more microRNAmimics may precede or follow administration of the other cancertherapeutic agent by intervals ranging from minutes to weeks. Inembodiments where the other cancer therapeutic agent and one or moremicroRNA mimics are applied separately to the subject, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the cancer therapeuticagent and the one or more microRNA mimics would still be able to exertan advantageously combined effect on the tumor cell.

Further pharmacological therapeutic agents and methods ofadministration, dosages, etc., are well known to those of skill in theart (see, for example, the “Physicians Desk Reference,” Klaasen's “ThePharmacological Basis of Therapeutics,” “Remington's PharmaceuticalSciences,” and “The Merck Index, Eleventh Edition,” incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention. They are not meant to limit the inventionin any fashion. One skilled in the art will appreciate that theinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well any objects, ends and advantagesinherent herein. The present examples (along with the methods describedherein) are presently representative of preferred embodiments. They areexemplary, and are not intended as limitations on the scope of theinvention. Variations and other uses which are encompassed within thespirit of the invention as defined by the scope of the claims will occurto those skilled in the art.

Example 1 Discovery of the Reduced Expression of the miR-15/107 Familyin MPM

MicroRNAs have important roles in cancer development and theirexpression is dysregulated in tumors, including MPM. The inventorsidentified changes in the miR-16 expression in MPM cell lines and asmall set of tumor samples, and further assessed expression of theentire miR-15/107 family of related microRNAs in MPM cell lines and alarger set of tumor specimens.

A panel of 7 MPM cell lines was compared with a mesothelial cell lineMeT-5A. Cells were obtained from ATCC(H28, H2052, H2452, H226 andMSTO-211H) and collaborators (MM05 and Ren) and cultured in therecommended medium at 37° C. with 5% CO₂. Total RNA was isolated fromcells (grown to 80% confluence) using TriZOL® (Life Technologies). Sixtytumor specimens consisted of formalin-fixed, paraffin-embedded blocks.Tumor microRNA expression was compared with 23 formalin-fixed samples ofnormal pleural tissue. To isolate RNA from tumor specimens, anexperienced pathologist marked tumor content on H&E stained slides fromeach block which were used as a guide for laser-capture microdissectionto enrich tumor content. Briefly, samples were mounted onto membraneslides (Zeiss), and LCM was carried out using the PALM system (Zeiss).Captured tissue was collected in adhesive collection tubes, anddeparaffinisation was performed in xylene. RNA was extracted from thecaptured tumor specimens and the normal tissues using the FFPE RNeasyMini kit (Qiagen) according to the manufacturer's instructions. RNA fromcell line and tissue samples was quantified using a Nanophotometer withreadings at 260 and 280 nm. For both cell line and tumor samples,reverse transcription (RT) used microRNA-specific stem-loop primers(Life Technologies). 100 ng total RNA was used as template in the RTreaction, also including 4 μl RT primer mix (with up to 10microRNA-specific RT primers multiplexed in an equimolar mix), with thereaction carried out following the manufacturer's instructions in avolume of 10 μl. The resultant complementary DNA (cDNA) was diluted bythe addition of 57.8 μl water, and from this dilution, 2.25 μl cDNA wasadded as template to the qPCR reaction. The qPCR further containedmicroRNA-specific TaqMan primers/probes and TaqMan GeneExpressionMastermix (both Life Technologies) with a total reaction volume of 10μl. The reactions were set up manually and run in duplicate on a Mx3000Preal-time PCR machine (Stratagene) with 10 min enzyme activation at 95°C. followed by 40 cycles of 15 sec denaturing at 95° C. and 60 secannealing/elongation at 60° C. Cq (quantification cycle) values weredetermined applying adaptive-baseline and background-based thresholdalgorithms using the MxPro software. Analysis of the qPCR results wasperformed using the 2^(−ΔΔCq) method (as described by Livak et al), andincluded normalization to RNU6B expression levels. Expression in eachsample was calculated relative to the average expression of controls.

Of the ten microRNAs in the miR-15/107 family, 6 (miR-16, miR-15a,miR-15b, miR-195, miR-103 and miR-107) were detected in all samples, and4 (miR-424, miR-497, miR-503 and miR-646) were undetected. In celllines, an average 2- to 5-fold downregulation of all six detectablemicroRNAs was observed as compared to expression in immortalized normalmesothelial cells. In tumors, miR-16, miR-15a, miR-15b and miR-195 weredownregulated by on average 8- to 25-fold, whereas miR-103 and miR-107were downregulated by 4- to 6-fold, when compared with levels of thesemicroRNAs in normal pleura.

Example 2 Reduction of miR-15/107 Family Expression Upon Exposure of MPMCells to Chrysotile Asbestos Fibres

Asbestos is the etiological agent in MPM, but effects on microRNAexpression are unknown. Changes observed following asbestos exposurewould suggest a causative role in MPM biology. Previous work in thefield has focused on the effects on cells of acute exposure to cytotoxicconcentrations of asbestos fibres. While changes in gene expression areobserved in these cases, the predominant result of such treatment is acombination apoptosis and necrosis leading to extensive cytotoxicity andcell death.

In order to identify the physiologically relevant effects of asbestosexposure on microRNA expression in mesothelial cells, MeT-5A cells werecontinuously exposed to chrysotile asbestos fibres. MeT-5A cells weregrown in recommended culture conditions in the presence of 0, 0.1 or 1μg chrysotile asbestos fibres continuously for 3 months. At theindicated time points, cells were harvested and RNA isolated formicroRNA expression measurements. RNA isolation and RT-qPCR was carriedas described in Example 1. Levels of microRNA were normalized to U6expression and are expressed relative to the normalized expression inuntreated cells. FIG. 2 demonstrates that continuous exposure toasbestos at 1 μM leads to decreased expression of miR-16, miR-15b,miR-195, miR-103 and miR-107 at all time points, and decreases inmiR-15a expression from 70 days on. These results are the first to linkasbestos exposure to changes in microRNA expression in general, and thefirst to analyze changes in gene expression related to long-termasbestos exposure. They provide a direct link between asbestos exposureand miR-15/107 family expression, and thus suggest that these may beearly and important changes in MPM progression.

Example 3 Replacing miR-15/107 Family Leads to Growth Inhibition of MPMCells In Vitro

In order to determine the effects of restoring miR-15/107 family memberson MPM cell growth, MPM cell lines were transfected with microRNA mimicsand the effect on cell growth measured. Mimics consisted ofdouble-stranded RNAs corresponding to the sequence of mature miR-16,miR-15a or miR-15, or a synthetic sequence corresponding to theconsensus sequence of the miR-15/107 family, and were provided asHPLC-purified lyophilized duplexes (Shanghai GenePharma). Mimics werere-suspended in water at a concentration of 20 μM. These were thenreverse transfected into cells using Lipofectamine RNAiMAX (‘LRM’, LifeTechnologies) at the indicated concentrations. First, lipoplexes weregenerated by mixing the appropriate concentration of mimic in serum-freemedium with an equal volume of a 1% solution of LRM in serum-freemedium, and incubating for 20 to 120 minutes at room temperature.Lipoplexes were distributed to replicate multiwell plates and cells insuspension (medium containing 10% FCS) were added to the lipoplex mix ineach well such that the final density of cells was 7500/cm².Transfection was allowed to proceed for 24 hours after which medium wasreplaced with fresh medium containing 10% FCS and cells were furtherincubated at 37° C. until harvest. Thereafter, replicate plates wereharvested at 48, 72, 96 and 120 hours post-transfection, which involvedremoving medium and freezing plates at −80° C. At the conclusion of theexperiment, plates were thawed and 150 μl lysis buffer containing 0.01%SYBR Green added to each well to measure DNA content. After incubationovernight in the dark, DNA content was quantified by measuringfluorescence in a Fluostar Optima fluorimeter, set at excitation of 485nm and emission 535 nm. Total fluorescence (i.e. DNA per well) in thisassay displays a linear relationship with cell number, allowing it todetermine proliferation.

Following transfection with microRNA mimics corresponding in sequence tomiR-15a, miR-15b, or miR-16 there was a dose- and time-dependentdecrease in proliferation in 4 MPM cell lines (H28, MM05, H2052 andMSTO). There was no effect of these treatments on the normal mesothelialcell line MeT-5A. Therefore, restoring microRNA expression and thuscontrol of target gene expression resulted specifically in theinhibition of proliferation of MPM cells.

To investigate further the effects of the miR-15/107 family on MPMcells, mimics were designed that correspond to the consensus sequence ofthe family. The consensus mimics appear below.

TABLE 1 Mimics Sense (passenger) Antisense (Guide) con15/107.1mCmGmCmAAACCAUUAUGUGCUmGmCmUmA UAGCAGCACAUAAUGGUUUGCG (SEQ ID NO: 15)(SEQ ID NO: 11) con15/107.2 mUmCmCmGCAAACCAUUAUGUGCUmGmCmUmAUAGCAGCACAUAAUGGUUUGCGGA (SEQ ID NO: 16) (SEQ ID NO: 12) con15/107.3mCmGmCmAAACCAUUAUGUGCUmGmCmUmA UAGCAGCACAUAAUGGUUUGCU (SEQ ID NO: 17)(SEQ ID NO: 13) con15/107.4 mCmGmCmAAACCAUACUGUGCUmGmCmUmAUAGCAGCACAGUAUGGUUUGCG (SEQ ID NO: 18) (SEQ ID NO: 14)

These consensus sequences varied depending on the number of microRNAsincluded in the alignment, varying from the miR-15 family only(con15-107.1) to the entire 15/107 family (con15-107.2 to 4). Theconsensus length in con15-107.3 was increased to account for the longermature sequence of some microRNAs. These 4 consensus microRNAs were thentransfected into MSTO cells at varying concentrations and the effect oncell growth assessed as above. FIG. 4 shows that all 4 mimics based onthe miR-15/107 consensus sequence were more growth inhibitory than thenative miR-16 sequence. This suggests that the consensus sequences aremore promising therapeutic candidates than the natural miR-16.

Example 4 Transfection with miR-16 Downregulates Target Genes

MicroRNAs are responsible for post-transcriptional gene regulation.While the primary mechanism of action of microRNAs is via inhibition oftranslation of mRNA into protein, this frequently leads todestabilization of target mRNA. Therefore, the ability of a microRNA toregulate gene expression of predicted targets can be measured byanalyzing target mRNA levels following modulation of the microRNA ofinterest. Targets regulated in this way are then candidates for genesinvolved in the phenotypic effects observed following microRNA mimictransfection. Those genes also downregulated at the protein level areconsidered more likely to be bona fide targets of the microRNA.

To link effects of mimic transfection on cell biology of MPM cells,expression of 24 candidate miR-16 target genes was measured in MPM cellstreated with miR-16 mimic. H28 and MSTO cells were reverse transfectedin 6-well plates, with miR-16 mimic or control (each 5 nM) as per themethod described for Example 3. After 48 h transfection, RNA wasisolated using TriZOL and quantified using a nanophotometer (Implen,Munich, Germany). From replicate wells, protein was isolated andquantified by Pierce BCA Protein assay (Thermo Fisher Scientific).Synthesis of cDNA used 250 ng RNA as template, with a mix of randomoligos and oligodT as primers. This cDNA was then used as template in 10μl qPCR reactions with primers specific for the predicted targets ofmiR-16, using Brilliant II SYBR green chemistry (Agilent Technologies)mix as per manufacturer's instructions, and the reactions were run on anMX3000P real time PCR machine (Agilent Technologies). The qPCR resultswere analyzed by the ΔΔCt method, whereby target gene expression wasnormalized to expression of 18S, and results from mimic-transfectedcells expressed relative to the normalized expression of targets incontrol-transfected cells. These results demonstrated a down-regulationin 24 targets ranging from 1.2 to 4-fold. On the protein level,expression of CCND1 and Bcl-2 were analyzed by western blot. Protein (20μg) was separated on a 10% precast polyacrylamide gel (Mini Protein TGXPrecast Gels, Biorad) and transferred to PVDF membranes using the BioradTrans-Blot Turbo Transfer System (Biorad, NSW, Australia). Membraneswere blocked using milk powder then probed with target specificantibodies (Bcl-2; CCND1), followed by detection with a rabbit or mousespecific secondary antibody (all antibodies from Cell Signaling Inc).Chemiluminesence (Supersignal West Femto Maximum Sensitivity substratekit, Thermo Fisher Scientific) was used to detect the presence of theprotein and was measured using a Kodak Geologic 2200 imaging system.Expression of beta-actin (β-actin) was included to control for equalprotein loading. Protein expression of both CCND1 and Bcl-2 weresignificantly down-regulated in miR-16 treated cells compared withcontrols. Together, these changes in mRNA and protein expression showthat the observed phenotypic effects of miR-16 mimic transfection arerelated to genes involved in proliferation and altered apoptoticresponse.

Example 5 Effects of miR 16 on Gemcitabine and Pemetrexed Toxicity inMPM Cells

MPM is considered resistant to chemotherapy, and this resistance isbelieved to relate to changes in apoptotic responses of the tumor cells.As many of the predicted targets of the miR-15/107 family are genesrelated to these processes, one might predict that microRNA mimics wouldsensitize MPM cells to chemotherapy drugs. This was tested for thecombination of miR-16 and the drugs gemcitabine and pemetrexed.

To test the effect of restoring miR-16 expression on drug toxicity, thenormal mesothelial cell line MeT-5A (A, D), and two MPM lines—MM05 (B,E) and MSTO-211H (C, F)—were transfected with 1 or 5 nM miR-16 (closedsymbols) or control mimic (closed symbols) in 96-well plates asdescribed in Example 3. Thereafter, medium was replaced after 24 hourswith medium containing a serial dilution of pemetrexed (1.95 to 500 nM)or gemcitabine (0.625 to 160 nM) with each concentration assayed intriplicate. After 72 hours, plates were harvested and DNA contentmeasured as described in Example 3. The growth of cells exposed to drugwas normalized to untreated cells, and the concentration inhibitinggrowth by 50% (IC₅₀ value) was determined. The effects of miR-16 on drugsensitivity was determined by comparing IC₅₀ values in mimic and controltransfected cells. This demonstrated a dose-dependent 2- to 5-foldsensitization of MM05 (B, E) and MSTO-211H (C, F) cells to both drugs,but no effect on normal MeT-5A cells. (A, D).

Example 6 Effects of miR-16 Replacement in MPM In Vivo, Delivered asVectEDVmiR-16

The growth (and other) inhibitory effects observed upon restoration oflost expression of tumor suppressor microRNAs in cancer suggests thatthey represent novel therapeutic targets. In order to investigate thispossibility, this must be tested in pre-clinical mouse models. In orderto replicate these in vitro effects in the in vivo situation, however,the microRNA mimics must overcome hurdles limiting delivery to the cellswithin the tumor where they have their therapeutic effect. Here thedelivery of miR-16 mimic was delivered using a targeted minicellapproach. Minicells are nanoparticles derived from asynchronous divisionof bacteria, as disclosed above in reference to U.S. Patent Pub. No.2011/0111041.

In vivo efficacy of miR-16 restoration was evaluated in a subcutaneoushuman xenograft model of MPM in nude mice. Athymic (nu/nu) mice (4-6weeks old) were purchased from the Animal Resources Centre (PerthWestern Australia) and all animal experiments were approved by theSydney Local Health Districts Animal Ethics Committee, Concord and RPAH.MSTO cells were cultured and 1.5×10⁶ cells in 50 μl serum-free mediatogether with 50 μl growth factor reduced matrigel (BD Biosciences) andinjected subcutaneously between the shoulder blades. Tumor volume (mm³)was determined by measuring length (l) and width (w) and calculatingvolume (V=lw²/2) as described on the indicated days. Experimental andcontrol treatments were carried out once the tumor volumes were onaverage 100 mm³, at which time the tumor mass was clearly palpable andvascularized, as determined following excision and histologicalexamination of tumors. Mice were randomized to different groups beforestarting the various treatments. All tumor volume-measurements wereperformed by an investigator blinded to the treatments administered.

Two experiments were carried out. In the first experiment, mice weretreated on the indicated days with indicated dose of 1×10⁹ miR-16- orcontrol-containing minicells 1, 2 or 4 times per week. The tumors in thecontrol mice (treated with saline or empty minicells) increased from 100to 400 mm³ in the course of the experiment. Tumors in those micereceiving miR-16 mimic grew more slowly, and effects were dependent onnumber of treatments. Tumor-bearing mice receiving 1 dose per week hadtumors that grew more slowly than those in control-treated mice, andthis inhibition of growth was maintained until day 30, at which pointthe size of these tumors was similar to that of controls. Mice receiving2 doses per week had tumors that increased in size to approximately 200mm³ by the end of the experiment on day 33, and were considerablysmaller than those in mice receiving control minicells throughout. Micetreated 4 times per week had tumors that did not increase in size forthe first week following the initial administration of the miR-16 mimic.By the end of the experiment, these tumors increased in size to only 170mm3, corresponding to a 75% inhibition of growth when compared withcontrols.

In the second experiment, mice were treated 4 times per week with a doseof 2×10⁹ miR-16 or control-loaded minicells. In this experiment, tumorsin the mice treated with control minicells grew at a comparable rate tothose in the first experiment that received half the dose. In micereceiving the increased dose of miR-16, there was a marked increase inanti-tumor effect of the miR-16 mimic. In the initial phase followingtreatment, the volume of these tumors decreased from the 100 mm³starting point. From there, tumor volume remained around 80 mm³ anddespite a slight increase following cessation of treatment on day 26 andthe end of the experiment on day 29, remained below 100 mm³. Thiscorresponds to a complete inhibition of tumor growth in thesemiR-16-treated animals compared with saline and control-treated groups.

The inhibition of growth of MSTO-211H-derived xenograft tumors observedin mice following administration of ^(Vect)EDV_(miR-16) is exceptionallystrong and exceeds the inhibition observed in vitro in cultures of thesame (and other) MPM cells (compare FIG. 3 with FIG. 7). This isremarkable considering the fact that in vitro, >95% of cells aretransfected with the miR mimic, whereas in vivo, the number of tumorcells receiving the mimic is likely to be ≦10%. It is likely that theobserved effect is caused by the inhibitory effects of miR-16 (and otherfamily members) on endothelial cells, thereby effectively targeting bothtumor cells and stromal cells involved in angiogenesis that is requiredfor tumor growth. It is noteworthy that the growth inhibitory effects ofthe mimic treatment in the subcutaneous xenograft model are greater thaneffects reported for other systemic treatments in the same model.

The present invention is well adapted to attain the ends and advantagesmentioned as well as those that are inherent therein. The particularembodiments disclosed above are illustrative only, as the presentinvention may be modified and practiced in different but equivalentmanners apparent to those skilled in the art having the benefit of theteachings herein. Furthermore, no limitations are intended to thedetails of construction or design herein shown, other than as describedin the claims below. It is therefore evident that the particularillustrative embodiments disclosed above may be altered or modified andall such variations are considered within the scope and spirit of thepresent invention.

The invention claimed is:
 1. A double-stranded microRNA mimic for thetreatment of malignant pleural mesothelioma (MPM), wherein the microRNAmimic comprises: (a) a consensus sequence of the miR-15/107 family,wherein the consensus sequence comprises a sequence selected from thegroup consisting of SEQ ID NOS: 11-14; and (b) a passenger strand.
 2. Adouble-stranded microRNA mimic for the treatment of malignant pleuralmesothelioma (MPM), wherein the microRNA mimic comprises: (a) aconsensus sequence of the miR-15/107 family, wherein the consensussequence contains AGCAGC at positions 2-7 at the 5′ end but is differentfrom the natural sequence of any miR-15/107 family member; and (b) apassenger strand, wherein the passenger strand comprises a sequenceselected from the group consisting of SEQ ID NOS: 15-18.
 3. Thedouble-stranded microRNA mimic of claim 1 or 2, wherein the microRNAmimic comprises one or more nucleotides that are modified.
 4. Adouble-stranded microRNA mimic for the treatment of malignant pleuralmesothelioma (MPM), wherein the microRNA mimic comprises: (a) aconsensus sequence of the miR-15/107 family, wherein the consensussequence comprises a sequence that differs from any one of SEQ ID NOS:11-14 by one or more nucleotides that are modified therefrom; and (b) apassenger strand.
 5. The double-stranded microRNA mimic of claim 2,wherein the passenger strand is inactivated by chemical modification. 6.The double-stranded microRNA mimic of claim 1, wherein the passengerstrand is inactivated by chemical modification.
 7. The double-strandedmicroRNA mimic of claim 4, wherein the passenger strand is inactivatedby chemical modification.